Bottom Line:
Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum.Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco.Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.

Affiliation: Department of Biochemistry, University of Nebraska, Lincoln, NE 68588, USA, Deparment of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA and Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE 68588, USA.

ABSTRACTThe type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5' coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.

gkt780-F6: Gene constructs in a binary vector used in transformation of immature sorghum embryos to test for Cas9/sgRNA activity. A single binary vector was designed and constructed to contain (A) a chimera of a wild-type GFP gene and a neomycin phosotransferase gene, (B) a non-functional mutant DsRED2 gene, (C) a Cas9 gene codon-optimized for expression in maize, (D) a sgRNA gene driven by a rice U6 promoter and targeting the mutant DsRED2 gene.

Mentions:
In a strategy similar to that used above with Arabidopsis and tobacco, an A. tumefaciens binary vector, Y158, was designed and constructed that carries four independent genes—an out-of-frame red fluorecence protein gene (DsRED2) (the target for Cas9-sgRNA cleavage and mutagenesis), a synthetic Cas9 gene codon optimized for expression in monocots, a U6 promoter-driven sgRNA gene and a GFP-NptII fusion visual/selectable marker gene (Figure 6). The maize ubiquitin 1 promoter/intron combination expresses a DsRED2 coding region with a nopaline synthase (NOS) 3′ end. The DsRED2 is intentionally designed to be out of frame and contains a target for the Cas9/sgRNA complex near the beginning of the DsRED2 coding region. Downstream of the DsRED2 chimeric gene is a rice Actin 1 promoter/intron combination expressing a synthetic Cas9 coding region. The octopine synthase 3′ polyadenylation region is downstream of the Cas9 coding region. A rice U6 promoter is used to express a U6 transcript as a guide RNA with a targeting sequence at its 5′ end. These three gene cassettes (DsRED2 target, Cas9 and sgRNA) are contained within the T-DNA region of a pVS1-derived binary vector. The T-DNA region of this binary vector also contains a GFP-NptII fusion gene expressed from the CaMV 35 S promoter and maize hsp70 intron. The expression of the GFP-NptII fusion protein allows transformed cells to be identified by their GFP expression.Figure 6.

gkt780-F6: Gene constructs in a binary vector used in transformation of immature sorghum embryos to test for Cas9/sgRNA activity. A single binary vector was designed and constructed to contain (A) a chimera of a wild-type GFP gene and a neomycin phosotransferase gene, (B) a non-functional mutant DsRED2 gene, (C) a Cas9 gene codon-optimized for expression in maize, (D) a sgRNA gene driven by a rice U6 promoter and targeting the mutant DsRED2 gene.

Mentions:
In a strategy similar to that used above with Arabidopsis and tobacco, an A. tumefaciens binary vector, Y158, was designed and constructed that carries four independent genes—an out-of-frame red fluorecence protein gene (DsRED2) (the target for Cas9-sgRNA cleavage and mutagenesis), a synthetic Cas9 gene codon optimized for expression in monocots, a U6 promoter-driven sgRNA gene and a GFP-NptII fusion visual/selectable marker gene (Figure 6). The maize ubiquitin 1 promoter/intron combination expresses a DsRED2 coding region with a nopaline synthase (NOS) 3′ end. The DsRED2 is intentionally designed to be out of frame and contains a target for the Cas9/sgRNA complex near the beginning of the DsRED2 coding region. Downstream of the DsRED2 chimeric gene is a rice Actin 1 promoter/intron combination expressing a synthetic Cas9 coding region. The octopine synthase 3′ polyadenylation region is downstream of the Cas9 coding region. A rice U6 promoter is used to express a U6 transcript as a guide RNA with a targeting sequence at its 5′ end. These three gene cassettes (DsRED2 target, Cas9 and sgRNA) are contained within the T-DNA region of a pVS1-derived binary vector. The T-DNA region of this binary vector also contains a GFP-NptII fusion gene expressed from the CaMV 35 S promoter and maize hsp70 intron. The expression of the GFP-NptII fusion protein allows transformed cells to be identified by their GFP expression.Figure 6.

Bottom Line:
Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum.Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco.Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.

Affiliation:
Department of Biochemistry, University of Nebraska, Lincoln, NE 68588, USA, Deparment of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA and Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE 68588, USA.

ABSTRACTThe type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5' coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.